| BackgroundHepatocellular carcinoma (HCC) is a common type of cancer worldwide, and is the third most common cause of cancer-associated mortality. The therapeutic effects of current treatment strategies for patients with HCC remain unsatisfactory, thus it is important to develop novel methods of treatment for this disease. B cell lymphoma (Bcl)-2, Bcl-extra large (xL) and Bcl-2-like-2 (w) are the anti-apoptotic protein members of the Bcl-2 family in mammalian cells, which contain four BH domains (BH1, BH2, BH3 and BH4). The Bcl-2 anti-apop-totic proteins have been reported to prevent mitochondrial outer membrane permeabilization and repress apoptosis under stress. The expression of Bcl-2 anti-apoptotic proteins is often increased in numerous types of tumor tissue, which is commonly associated with treatment resistance Previous studies have found that the levels of Bcl-2 anti-apoptotic proteins are closely associated with the pathological grade and survival rate of patients with,HCC. Therefore, the targeting of Bcl-2 anti-apoptotic proteins may be a candidate for the treatment of patients with HCC. ABT-737 is an inhibitor of Bcl-2, Bcl-xL and Bcl-w, which is currently in phase I clinical trials for patients with leukemia. It has been previously reported that ABT-737 also has antitumor effects in HCC cell lines. However, certain pro-survival signaling pathways in HCC cells are often activated upon ABT-737 treatment, which attenuates the antitumor effect of ABT-737. Therefore, novel treatment strategies require investigation in order to improve the effcacy of ABT-737. Curcumin, also known as diferuloylmethane, is obtained from Curcuma longa, and is regarded as a potent anticancer drug in various types of tumor, including HCC. Previous studies have demonstrated that curcumin is able to effectively inhibit the proliferation of HCC cells in vitro and in vivo. In addition, curcumin signifcantly enhances the antitumor effects of certain traditional chemotherapeutic drugs and molecular-targeted drugs. However, the synergistic effect of curcumin and ABT-737 remains to be fully elucidated.ObjectThe present study aimed to investigate the antitumor effects of combination therapy of ABT-737 with curcumin on HepG2 cells. Whether curcumin enhances the antitumor effect of ABT-737 via the induction of apoptosis in HepG2 cells was investigated, and the potential involvement of the reactive oxygen species (ROS)-apoptosis signal-regulating kinase 1 (ASK1)-c-Jun N-terminal kinase (JNK) pathway was examined. The study on the antitumor effects by curcumin-enhanced ABT-737, is intended to provide experimental bases and references of the further applications of curcumin and ABT-737.Methods(1) Human hepatoma cell line HEPG2 were randomly divided into 3 groups:① ABT-737 concentration gradient group:respectively 0 μM,1 μM,5 μM,10 μM,15 μM concentration gradient acting on HEPG2 cell; ② curcumin concentration gradient group:respectively 0μM,2 μM,4 μM,6μM,8 μM concentration gradient acting on HEPG2 cell;③) combined treatment group:Select curcumin concentration with minimal impact and 0 μM,1μM,5 μM,10μM ABT-737 combined effects on HEPG2 cells.The death rate of HEPG2 cells was determined by Trypan Blue stain assay. The cell proliferation was tested by CCK-8 assay.(2) We randomly divided HEPG2 cells into three groups, namely ① DMSO solvent control group; ② single drug group:10 μM ABT-737 group and 2 μM curcumin group;③) combination group:ABT-73710 μM+2 μM curcumin compatibility. We have used Hoechst 33258 staining to detect apoptotic in each group; we also detected the Caspase-3 activity by Caspase 3 activity detection kits. Bcl-2、 Mcl-1、PARP、C-PARP、C-caspase-3 protein expression were detected by western blot. We have used reactive oxygen species assay kit/ROS assay kit to detect the level of ROS.(3) HEPG2 cells were randomly divided into three groups:the first group was divided into 10μM ABT-737+2μM curcumin group and 10μM ABT-737+2μM curcumin group+10 μNAC group (ROS inhibitor); the second group was divided into 10 μM ABT-737+2μcurcumin group and 10μABT-737+2μM curcumin+ SP600125 (JNK inhibitor); the third group was divided into control group that transfected with empty plasmid vector, followed by 10 μM ABT-737+2 μM curcumin-treated cells and ASK1 siRNA group that transfected ASK1 siRNA, followed by 10 μM ABT-737+2 μM curcumin-treated cells. We detected the apoptosis and Caspase-3 activity by Hoechst 33258 staining experiments, Annexin V-FITC/PI double staining and apoptosis Caspase-3 activity assay kit. Western blot was used to detect p-JNK1 and p-JNK2, JNK1 and JNK2 protein expression in 1%DMSO control,2μM curcumin,10 μM ABT-737 and 10μM ABT-737+2 μM curcumin group; and then we detected p-JNK1 and p-JNK2, JNK1 and JNK2 protein expression by western blot in the first group. Annexin V-FITC/PI double staining was used to detected the apoptosis in the second group. Trypan blue exclusion assay was used to detected the apoptosis in the third group.Results(1) ABT-737 and curcumin can significantly inhibit the HEPG2 cells viability and in a dose-dependent manner (F=594.692, P=.000), (F=422.147, P=.000); there was cytotoxicity in HEPG2 cells after effected with ABT-737 and curcumin and in a dose-dependent manner (F=1127.812, P=.000), (F=1539.563, P=.000).In order to exclude interference in the experiments, we had chosed the 2 μM curcumin that has no significant effect in cell HEPG2. when we respectively used different concentrations of ABT-737 and 2 μM curcumin compatible to effect HEPG2 cells, the result showed the cell viability inhibition rate and cell mortality was significantly higher than the corresponding concentration of ABT-737 when used alone, and the cell viability inhibition rate and cell mortality was the highest when used 10 μM ABT 737+2μM curcumin, and in a dose-dependent manner (F=799.555, P=.000), (F=1372.177, P=.000)(2) Hoechst 33258 assay showed that the number of apoptotic cells in 10 μM ABT-737+2μM curcumin group was significantly higher than it in 2μM curcumin group and 10 μM ABT-737 group. Caspase-3 activity assay showed that caspase-3 activity in 2 μM curcumin group was no significant changes, but caspase-3 activity in 10 μM ABT-737 group was slightly changes; compared with 10 μM ABT-737 group, the caspase-3 activity in 10μM ABT-737+2 μM curcumin group was significantly enhanced (2.250±0.132,1.524±0.086, F=251.578, P=.000). Western blot results showed that the expression of C-caspase-3 protein in 10 μABT-737+2 μM curcumin group was significantly higher than it in 10 μM ABT-737 group (0.841±0.055,0.544±0.047, F=88.286, P=.000). ROS drtection results showed the ROS in 10 μM ABT-737+2 μM curcumin group was significantly higher than it in 10 μM ABT-737 group (2.257±0.088,1.231±0.068, F=566.789, P=000)(3) When added NAC (ROS inhibitor) in 2μM curcumin+10 μM ATB-737 group, the number of apoptosis cells (26.417±2.714,15.540±1.587, t=14.640, P=000) and the Caspase-3 activity (2.276±0.025,1.518±0.053, t=38.688, P=.000) were significantly decreased. Western blot results showed that the expression of p-JNK protein in 2μM curcumin+10μM ATB-737 group was higher than it in 10 μM ABT-737 group (0.914±0.043,0.714±0.034, F=134.864, P=000; 0.907±0.078, 0.710±0.039, F=71.176, P=.002); when added the NAC in 2μM curcumin+10μM ATB-737 group, the expression of p-JNK protein was significantly reduced (0.717±0.093,0.924±0.079,t=2.942,P=0.042;0.767±0.078,0.918±0.039,t=4.121, P=0.015). Annexin V-FITC/PI double staining showed that when added the SP600125 (JNK inhibitor) in 2μM curcumin+10μM ATB-737 group, the number of apoptotic cells were significantly reduced (25.326±2.122,14.450±1.625, t=14.983, P=.000). After silenced ASK1 in HEPG2 cells in 2μM curcumin+10 μM ATB-737 group, the rate of cell death was significantly reduced (39.050±5.930,26.657±3.104, t=18.578, P=.000)ConclusionCurcumin can promoted the ABT-737-inducted apoptosis of HEPG2 cells by increasing the level of ROS and then activating ASK 1-JNK signaling pathway. |
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